Hormone-dependent malignancies of the prostate and breast are leading causes of cancer incidence and death in the Western world. For example, prostate cancer (PC) is the second most common cancer in American men and is responsible for about 11% of all cancer related deaths (Jemal et al., 2010, Cancer J. Clin. 60:277; Altekruse et al., Eds., SEER Cancer Statistics Review, 1975-2007; National Cancer Institute: Bethesda, Md., 2010).
Since the pioneering studies of Charles Huggins, hormonal ablative therapy of these diseases has become standard practice (Huggins & Hodges, 1941, Cancer Res. 1:293-397; Huggins, 1954, J. Natl. Cancer Inst. 15:1-25; Huggins & Yang, 1962, Science 137:257-62; Huggins, 1965, Cancer Res. 25:1163-67). Modern approaches include targeting steroid receptors in these tissues with androgen receptor antagonists (i.e., flutamide, also known as 2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-propanamide; Trachtenberg et al., 2002, Can. J. Urol. 3:240-45) or estrogen receptor antagonists (i.e., tamoxifen, also known as (Z)-2-[4-(1,2-diphenyl-but-1-enyl)phenoxy]-N,N-dimethyl-ethanamine; MacGregor & Jordan, 1998, Pharmacol. Rev. 50:1551-96). With the realization that agents that antagonize androgen and estrogen receptors in one tissue may act as agonists in another tissue, the accepted term for these agents are selective androgen receptor modulators (SARMs) and selective estrogen receptor modulators (SERMs), respectively. SARMs that target prostate cancer need only act as an androgen receptor antagonist in the prostate.
Prostate cancer is initially dependent on testicular androgens and is thus responsive to androgen ablation with surgical or chemical castration. The drug of choice for chemical castration is the luteinizing hormone-releasing hormone (LH-RH) agonist leuprolide (p-Pro-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt (SEQ ID NO:6); Leupron). Leuprolide inhibits the release of LH from the anterior pituitary and prevents Leydig cell testosterone biosynthesis. Supplementation of castration with blockade of androgen action in the prostate is common and may be achieved with an AR antagonist (bicalutamide, also known as N-[4-cyano-3-(trifluoromethyl) phenyl]-3-[(4-fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide) or by inhibition of type 1 5α-reductase (SRD5A 1) and type 2 5α-reductase (SRD5A2) with dutasteride ((5α,17β)-N-{2,5 bis(trifluoromethyl) phenyl}-3-oxo-4-azaandrost-1-ene-17-carboxamide). Bicalutamide is a relatively weak ligand for the AR and in castrate resistant prostate cancer (CRPC) this compound can even act as a weak agonist leading to the desire for better agents (Tran et al., 2009, Science 324:787-90).
In prostate cancer the therapeutic benefit of androgen deprivation therapy (ADT) is temporary and is often followed by recurrence of a more aggressive metastatic disease—CRPC. CRPC is characterized by elevated intratumoral androgen biosynthesis, increased androgen receptor (AR) signaling and expression of pro-survival genes despite castrate level circulating androgen concentrations (Knudsen & Scher, 2009, Clin. Cancer Res. 15:4792; Knudsen & Penning, 2010, Trends Endocrinol. Metab. 21:315; Locke et al., 2008, Cancer Res. 68:6407). The source of intratumoral androgens is likely dehydroepiandrosterone (DHEA) and/or 4-androstene-3,17-dione (Δ4-AD) from the adrenal, which is subsequently metabolized to testosterone and 5α-dihydrotestosterone (5α-DHT). The conversion of testosterone to 5α-DHT is catalyzed by 5α-reductase isoforms. While the use of 5α-reductase inhibitors in the treatment of CRPC is still under clinical investigation, chemoprevention trials of prostate cancer with both finasteride (a selective 5α-reductase type 2 inhibitor) and dutasteride (a combined 5α-reductase type 1 and type 2 inhibitor) have produced controversial outcomes (Andriole et al., 2010, N. Engl. J. Med. 362:1192-1202; Thompson et al., 2007, J. Clin. Oncol. 25:3076-81; Walsh, 2010, New Engl. J. Med. 362:1237-38).
Prostate tumor reappearance appears to be driven by adaptive intratumoral androgen synthesis that bypasses the effects of ADT (Attard et al., 2009, Cancer Res. 69:4937-40). This conclusion is supported by the success of the new drug abiraterone acetate ((3S,8R,9S,10R,13S,14S)-10,13-dimethyl-17-(pyridin-3-yl)-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol acetate; Johnson & Johnson) at arresting CRPC and reducing the size of bone metastases in ongoing phase II/III clinical trials (Attard et al., J. Clin. Oncol. 27:3742-48; Reid et al., 2010, J. Clin. Oncol. 28:1489-95). Approved by the FDA for the treatment of CRPC, abiraterone acetate is a steroidal P450 17α-hydroxylase/17,20-lyase (CYP17) inhibitor that blocks the conversion of pregnenolone to dehydroepiandrosterone (DHEA). It blocks this step in the adrenal, which is the major source of circulating DHEA, or in the prostate, if there is de novo steroidogenesis from cholesterol. One disadvantage is that CYP17 is high up in the steroidogenic pathway. Its inhibition in the adrenal diverts pregnenolone to form the mineralocorticoid desoxycorticosterone (DOC). In addition, CYP17 inhibition prevents the formation of cortisol, which feeds-back and inhibits the production of ACTH in the anterior pituitary. The combined effect is elevated DOC production and potentially life-threatening hypertension. Abiraterone is therefore co-administered with a glucocorticoid (hydrocortisone or prednisone) to suppress the hypothalamo-pituitary-adrenal axis. Chronic use of glucocorticoids can however lead to drug induced Cushing's syndrome, immunosuppression and osteoporosis. A second generation analog of abiraterone acetate is VN/124-1 or TOK-001, which is both a CYP17 inhibitor and an AR antagonist that targets the receptor for degradation (Vasaitis et al., 2008, Mol. Cancer Therap. 7:2348-57). VN/124-1 validates the concept that the androgen axes in CRPC may be effectively shut down by inhibiting local androgen biosynthesis and by blocking the AR.
Another approach to treatment has been to develop a more potent AR antagonist than bicalutamide, whose effects may be easily surmounted by intratumoral androgen synthesis, and acts as a weak androgen receptor agonist. MDV3100 (Medivation) also known as enzalutamide prevents both AR nuclear translocation and binding to DNA and is more potent than bicalutamide and does not exhibit agonist activity (Tran et al., 2009, Science 324:787-90). MDV3100 phase I/II clinical trials show that this agent reduces serum PSA, circulating tumor cells and causes radiographic stabilization of the disease (Scher et al., 2010, Lancet 375:1437-46). However, MDV3100 belongs to a class of anti-androgens that carry seizure risk, likely mediated via antagonism of the CNS-based GABAA receptor (Foster et al., 2010, Prostate 71:480-8). MDV3100 may ultimately be replaced by the use of its congener ARN-509 (Clegg et al., 2012, Cancer Res 72:1494-1503). MDV3100 has also been approved by the FDA for the clinical treatment of CRPC.
Steroid-target tissues produce steroid hormones locally (intracrine and paracrine formation) to maintain their growth (Labrie et al., 1995, Ann. Endocrinol. 56:23-29; Labrie et al., 2000, J. Mol. Endocrinol. 25:1-16). This is important in prostate and breast cancer, which are diseases of the aging male and female. In these individuals, the gonadal production of steroid hormones has been compromised (e.g. andropause and menopause). Enzymes important in the local production of androgens (e.g. type 2 5α-reductase) and estrogens (e.g. aromatase) have become the targets for inhibitor development to treat hormone dependent malignancies of the prostate and breast (Miller, 1996, Endocrine-Related Cancer 3:65-79; Gormley, 1996, Endocrine-Related Cancer 3:65-79). Effective agents include finasteride and exemestane (10,13-dimethyl-6-methylidene-7,8,9,10,11,12,13,14,15,16-decahydrocyclopenta[a] phenanthrene-3,17-dione), which are mechanism-based inactivators for type 2 5α-reductase and aromatase, respectively (Bull et al., 1996, J. Amer. Chem. Soc. 118:2359-65; Brodie et al., 1981, Steroids 38:693-702). One disadvantage to this approach is that aromatase inhibitors cause global decreases in active estrogens and can have unintended side effects (e.g. osteoporosis). Also such treatments can cause these malignancies to become refractory to hormone ablation.
The local levels of steroid hormones are also regulated by hydroxysteroid dehydrogenases (HSDs) (Penning, 1997, Endocrine Rev. 18:281-305). HSDs catalyze the NAD(P)(H) dependent oxidation or reduction of potent steroid hormones to their cognate inactive metabolites and vice-versa. For each steroid hormone there are pairs of HSDs that will either act as reductases or oxidases to modulate the potency of the hormone. For example, type 1 17β-HSD reduces E1 to E2 and is estrogenic, while type 2 and type 4 17β-HSD will oxidize E2 to E1 and attenuate estrogen action (Labrie et al., 2000, J. Mol. Endocrinol. 25:1-16; Labrie et al., 1997, Steroids 62:148-58; Labrie et al., 1995, Cell Biol. 14(10):849-61; Adamski et al., 1995, Biochem J. 311(Pt 2):437-43).
Aldo-keto reductases are a superfamily of 15 families of generally monomeric (37 kDa) cytosolic NAD(P)(H)dependent oxidoreductases that convert carbonyl groups to alcohols. Natural substrates for these enzymes include steroids, prostaglandins and lipid aldehydes (Hyndman et al., 2003, Chem-Biol. Inter. 143-144:621-31). All the HSDs in this superfamily are highly related in sequence (>67% identity) and comprise the AKR1 C family. The four human AKR1C isoforms (AKR1C1-SEQ ID NO:1; AKR1C2-SEQ ID NO:2; AKR1C3-SEQ ID NO:3; AKR1C4-SEQ ID NO:4) have been cloned and expressed, their enzymatic properties in vitro have been assigned, and their tissue specific expression patterns have been studied (Penning et al., 2000, Biochem. J. 351:67-77). Each of the four isoforms share >86% sequence identity but have different ratios of 3-, 17- and 20-ketosteroid reductase activities. AKR1C1 is a major 20α-HSD, AKR1C2 (type 3 3α-HSD and bile acid binding protein) is a major peripheral 3α-HSD, AKR1C3 is a peripheral 17β-HSD, and AKR 1 C4 (type 1 3αHSD/chlordecone reductase) is a hepatic specific 3α-HSD (Penning et al., 2000, Biochem. J. 351:67-77). These enzymes have the potential to regulate ligand occupancy of the PR, AR, and ER. This is referred to as the pre-receptor regulation of nuclear receptors (FIG. 1).
AKR1C3 catalyzes the NADPH dependent reduction of carbonyl moieties on substrates of importance to the pre-receptor regulation of signaling pathways involved in cell proliferation. The interconversion of a ketone group with a hydroxyl group on lipophilic ligands can drastically alter their affinity for their cognate receptors (FIG. 2). For example, AKR1C3 reduces the 17-position of Δ4-AD (a weak androgen) to form testosterone (a potent androgen) and of estrone (a weak estrogen) to form 17β-estradiol (a potent estrogen), leading to trans-activation of the androgen and estrogen receptors, respectively (Byrns et al., 2010, J. Steroid. Biochem. Mol. Biol. 118:177-87; Dufort et al., 1999, Endocrinology 140:568-74). Because of this activity, AKR1C3 is also known as type 5 17β-hydroxysteroid dehydrogenase. It can also act at the 20-position of progesterone and deoxycorticosterone, forming 20α-hydroxy metabolites with reduced affinities for the progesterone and mineralocorticoid receptors, respectively (Sharma et al., 2006, Mol. Cell. Endocrinol. 248:79-86). Finally, as prostaglandin (PG) F synthase, AKR1C3 catalyzes the stereo-specific reduction of PGH2 to PGF2α and of PGD2 to 9α,11β-PGF2 (Byrns et al., 2010, J. Steroid. Biochem. Mol. Biol. 118:177-87; Suzuki-Yamamoto et al., 1999, FEBS Lett. 462:335-40; Koda et al., 2004, Arch. Biochem. & Biophys. 424:128-36). In the absence of AKR1C3 activity, PGD2 spontaneously dehydrates and rearranges to form the PGJ2 prostanoids (Byrns et al., 2010, J. Steroid. Biochem. Mol. Biol. 118:177-87). The PGF2 isomers are pro-inflammatory and enhance proliferation, while the PGJ2 products, particularly 15-deoxy-Δ12,14-PGJ2 (15dPGJ2), are anti-inflammatory, promote differentiation, and are anti-neoplastic via several mechanisms (Jabbour et al., 2005, Endocrinology 146:4657-64; Ray et al., 2006, J. Immunol). 177:5068-76; Diers et al., 2010, Biochem. J. 430:69-78; Nakata et al., 2006, Mol. Cancer Therapeutics 1827-35; Butler et al., 2000, Cell Growth Differ. 11:49-61; Scher & Pillinger, 2009, J. Investig. Med. 57:703-08).
The products of reactions catalyzed by AKR1C3 promote tumor growth. It is therefore an important target for the prevention or treatment of both hormone-dependent and hormone-independent cancers. AKR1C3 likely contributes to the development of CRPC through the intratumoral formation of the active androgen testosterone (Penning et al., 2008, Mol. Cell. Endocrinol. 281:1-8). Transcript levels and measurement of testosterone: 5α-dihydrotestosterone (5α-DHT) ratios support the notion that there is a reprogramming of androgen dependence in castrate resistant disease that favors formation of testosterone by AKR1C3 (Montgomery et al., 2008, Cancer Res. 68:4447-54; Mostaghel et al., 2007, Cancer Res. 67:5033-41). Other studies have shown that there three pathways to the potent androgen 5α-DHT in prostate cancer, and AKR1C3 is required for each pathway. The first pathway known as the “classical pathway” involves the sequence: DHEA→Δ4-Aandrostene-3,17-dione (Δ4AD)→testosterone→5α-DHT. In this pathway AKR1C3 catalyzes the NADPH dependent reduction of Δ4-AD→testosterone. The second pathway is known as the “backdoor pathway” in which 5α-reduction occurs at the level of the pregnanes and by-passes DHEA and testosterone altogether (Auchus, 2004, Trends, Endocrinol. Metab. 15: 432-38). The critical conversions are: 5α-pregnane-3,20-dione (DHP)→allopregnanolone→androsterone→3α-androstanediol→5α-DHT. In this pathway, AKR1C3 catalyzes the NADPH dependent reduction of androsterone→3α-androstanediol. The third pathway known as the “alternative pathway” involves the sequence: DHEA→Δ4-AD→5α-androstane-3,17-dione→5α-DHT (Chang et al., 2011, Proc. Natl. Acad, sci. USA 108, 13728-33). In this pathway AKR1C3 catalyzes the NADPH dependent reduction of 5α-androstane-3,17-dione→5α-DHT. Thus AKR1C3 plays essential roles in the formation of the potent androgens that activate the AR, irrespective of the pathway responsible for their formation, FIG. 3.
In the breast, AKR1C3 catalyzes the reduction of Δ4-AD to testosterone, which can undergo aromatization to form 17β-estradiol. In addition, AKR1C3 also reduces estrone to 17β-estradiol. Consistent with these activities AKR1C3 has been shown to promote proliferation of MCF-7 hormone-dependent breast cancer cells (Byrns et al., 2010, J. Steroid. Biochem. Mol. Biol. 118:177-87).
In the endometrium, AKR1C3 could increase estrogen levels and decrease progesterone levels and thus promote endometrial cancer cell proliferation (Smuc & Rizner, 2009, Mol. Cell. Endocrinol. 301:74-82).
By increasing proliferative PGF2 isomers and decreasing anti-proliferative PGJ2 products, the prostaglandin F synthase activities of AKR1C3 have the potential to impact both hormone-dependent and hormone-independent cancers. In particular, prostaglandin metabolism by AKR1C3 has been shown to prevent differentiation of leukemia cells and AKR1C3 inhibition is being explored as a treatment for acute myelogenous leukemia (AML) (Khanim et al., 2009, PLoS One 4 e8147).
AKR1C3 is over-expressed across a wide variety of cancers, including those of prostate and breast, and its expression increases with tumor aggressiveness (Guise et al., Cancer Res. 70:1573-84; Lin et al., 2004, Steroids 69:795-801; Stanbrough et al., 2006, Cancer Res. 66:2815-25; Hofland et al., 2010, Cancer Res. 70:1256-64; Nakamura et al., 2005, Endocrine-Related Cancer 12:101-07; Jansson et al., 2006, Cancer Res. 66:11471-77).
AKR1C3-catalyzed reactions also play important roles in other physiological and pathological processes that may be targets for therapeutic intervention. Androgen production by AKR1C3 is likely involved in the development of benign prostatic hyperplasia (Ballinan et al., 2006, Mol. Endocrinol. 20:444-58). Increased estrogen and decreased progesterone receptor signaling due to increased AKR1C3 activity could contribute to endometriosis and dysmenorrhea (Smuc et al., 2009, Mol. Cell. Endocrinol. 301:59-64). PGF2 isomers formed by AKR1C3 stimulate smooth muscle contraction during parturition, while the AKR1C3 substrate progesterone prevents parturition, so AKR1C3 inhibitors may be useful as progestational agents (Breuiller-Fouche et al., Biol. Reprod. 83:155-62; Andersson et al., 2008, J. Clin. Endocrinol. Metab. 93:2366-74). Interestingly, indomethacin has proven effective at stopping premature parturition. This effect is thought to result from its inhibition of the prostaglandin G/H synthases (PGHS), but indomethacin also inhibits AKR1C3 in the same therapeutic dose range. Use of indomethacin for this purpose is limited due to side-effects in the fetus that likely stem from the inhibition of PGHS activities in developing organ systems (Loudon et al., 2003, Best Pract. Res. Clin. Obstet. Gynaecol. 17:731-44).
Because PGF2α is involved in preventing adipocyte differentiation and 15dPGJ2 promotes differentiation of adipocytes, inhibiting the prostaglandin F synthase activities of AKR1C3 may have beneficial effects in diabetes similar to those observed with PPARγ agonists (Reginato et al., 1998, J. Biol. Chem. 273:1855-58; Kliewer et al., 1995, Cell 83:813-19). AKR1C3 is also involved in the metabolism of other steroids as well as prostaglandins, suggesting that AKR1C3 may be used to treat other diseases. Currently, a non-selective AKR1C3 inhibitor, 6-medroxyprogesterone acetate, is used in early clinical trials to treat AML in Europe, based on a clearly defined role of AKR1C3 in regulating the differentiation of AML cells (Khanin et al., 2009, PLoS One e1847). A selective AKR1C3 could be a promising lead for the treatment of AML.
A specific inhibitor of AKR1C3 would be a valuable tool to better understand AKR1C3 and its contribution to normal physiology and disease. One challenge in the development of an AKR1C3 inhibitor is that three other closely related enzyme isoforms (AKR1C1, AKR1C2, and AKR1C4) are also involved in steroid hormone metabolism (Penning et al., 2000, Biochem. J. 351:67-77). AKR1C4 is liver specific, while AKR1C1 and AKR1C2 are widely expressed across tissue types, including prostate. Inhibition of these three isoforms, particularly AKR1C1 and AKR1C2, is not desirable in prostate cancer as they act primarily as 3-ketosteroid reductases towards androgens. For example, AKR1C1 reduces 5α-DHT to 3β-androstanediol (a pro-apoptotic ligand of estrogen receptor β) and AKR1C2 reduces 5α-DHT to 3α-androstanediol (a weak androgen). Inhibition of these activities would promote androgen dependent proliferative signaling in the prostate and would be counterproductive.
Twenty-two crystal structures of different AKR1C3 ternary complexes have been deposited into the Protein Data Bank (Table 1) (Qiu et al., 2007, J. Biol. Chem. 282:8368-79; Komoto et al., 2004, Biochemistry 43:2188-98; Lovering et al., 2004, Cancer Res. 64:1802-10; Qiu et al., 2004, Mol. Endocrinol. 18:1798-1807; Komoto et al., 2006, Biochemistry 45:1987-96; Bennett et al., 1997, Structure 5:799-812).
TABLE 1Available crystal structures of AKR1C3 in the Protein Data Bank.PDBIDLigandsReference1RYONADP+, PGD2Komoto et al., 2004,1RY8NADP+, rutinBiochemistry 43:2188-981S1PNADP+, 2-methyl-2,4-pentanediol, acetate ionLovering et al., 2004,1S1RNADP+, 2-methyl-2,4-pentanediol, acetate ionCancer Res. 64: 1802-101S2ANADP+, indomethacin pH 6.0, unknownatom or ion, dimethyl sulfoxide1S2CNADP+, flufenamic acid, dimethyl sulfoxide1XFONADP+, 4-androstene-3,17-dione, acetate ionQiu et al., 2004, Mol.Endocrinol. 18: 1798-18072F38NADP+, bimatoprostKomoto et al., 2006,Biochemistry 45: 1987-961ZQ5NADP+, EM1404, acetate ionQiu et al., 2007, J. Biol.2FGBNADP+, hexaethylene glycol, acetate ionChem. 282: 8368-793UWENADP+. 3-phenoxybenzoic acid, 1,2-ethanediolYusaatmadja et al, releasedMay 20123UG8NADP+, indomethacin pH 7.5, 1,2-ethandiolYusaatmadja et al, releasedMay 20123UGRNADP+, indomethacin pH 6.8, 1,2-ethanediolYusaatmadja et al, releasedMay 20123R58NADP+, Naproxen, 1,2-ethanediolYusaatmadja et al, releasedMay 2012UFYNADP+, R-Naproxen, 1,2-ethanediolYusaatmadja et al, releasedMay 2012R43NADP+, Mefenamic acid, 1,2-ethanediolYusaatmadja et al, releasedMay 20123R6INADP+, meclofenamic acid, 1,2-ethanediolYusaatmadja et al, releasedMay 20123R94NADP+. flurbiprofen, 1,2-ethanediolYusaatmadja et al, releasedMay 20123R8HNADP+, zomepirac, 1,2-ethanediolYusaatmadja et al, releasedMay 20123R8GNADP+, ibuprofen, 1,2-ethanediolYusaatmadja et al, releasedMay 20123R7MNADP+, sulindac, 1,2-ethanediolYusaatmadja et al, releasedMay 20124FAMNADP+ 3-(3,4-Dihydroisoquinolin-2(1H)-Turnbull et al., 2012ylsulfonyl)benzoic acids, 12-ethanediol
In the twenty two crystal structures of AKR1C3, the enzyme is complexed with the cofactor NADP+ and a second ligand. Close inspection of these structures reveals that the binding site for the second ligand is large and can be dissected into the following sub-sites: oxyanion site, steroid channel, and three sub-pockets named SP1, SP2, and SP3. One common feature of all twelve AKR1C3 structures is an occupied SP1 site (albeit to different degree), while the occupancy of other sites varies with different ligands. The occupancies of sub-sites in AKR1C3 by four inhibitors EM1404, bimatoprost, flufenamic acid, and indomethacin are depicted in FIGS. 5A-5D. Understanding the binding interactions between ligands and their sub-pockets may aid inhibitor design and synthesis.
The oxyanion site refers to the conserved site that anchors the oxyanion intermediate formed during the enzyme reaction and consists of the catalytic residues Y55, H117 and NADP+ in all AKR1C enzymes (Bennett et al., 1997, Structure 5:799-812). This position is often found occupied by the oxygen atom of a carboxylic acid, ketone, or hydroxyl group of a ligand. Strong hydrogen bonding interactions form between the occupant and Y55 and H117 (2.6 Å and 2.8 Å, respectively). The positioning of the carbonyl group of a substrate at the oxyanion site is believed to be essential for productive binding since the anchoring brings the carbonyl group in proximity to the cofactor, allowing the reaction to proceed (Bennett et al., 1997, Structure 5:799-812; Jin & Penning, 2006, Steroids 71:380-91; Jin et al., 2001, Biochemistry 40:10161-68). Similarly, it is believed that the binding of the carboxylate or ketone group of an NSAID at the oxyanion site explains the general inhibition of AKR1C enzymes by NSAIDs. For example, the carboxylic acid moiety of flufenamic acid is anchored at the oxyanion site of AKR1C3, while the bridge carbonyl group of indomethacin at pH 6.0 binds via an unidentified atom to the oxyanion site of the enzyme (FIGS. 5C and 5D). At pH 7.0, indomethacin rotates so that its crabxylate is now tethered to the oxyanion site. Interestingly, the oxyanion site does not appear to significantly contribute to the binding of EM1404 and bimatoprost (FIGS. 5A and 5B) in AKR1C3, as the site was occupied by an acetate ion and a water molecule, respectively in the crystal structures of the AKR1C3.NADP+.EM1404 and AKR1C3.NADP+.bimatoprost complexes (Komoto et al., 2006, Biochemistry 45:1987-96).
The steroid channel refers to the elongated open channel that is also conserved in all AKR1C enzymes (Bennett et al., 1997, Structure 5:799-812). W227 and L/V54 are important gate keepers for the steroid channel and determine the positional and stereochemical specificities of the steroid transforming activity of AKR1C enzymes (Jin et al., 2001, Biochem. 40:10161-68). Interestingly, the steroid channel does not appear to be an important inhibitor binding site for AKR1C3. With the exception of EM1404, which has its steroid ring structure partially occupying the steroid channel, this channel is left empty by bimatoprost, flufenamic acid, and indomethacin (FIGS. 5A-5D).
The SP1 sub-pocket is formed by residues S118, N167, F306, F311, and Y319 in AKR1C3, and is the only site that is occupied in all available crystal structures of this enzyme. As such, it accommodates the lactone moiety of EMI404 (FIG. 5A), the 12β-chain of PGD2, the 8α-chain of bimatoprost (FIG. 5B), or the —CF3 substituted B-ring of flufenamic acid (FIG. 5C). The p-chlorobenzoyl group of indomethacin also projects into this site, although in that structure a solvent molecule from the crystalization solution (dimethyl sulfoxide) was bound in the bottom of SP1 (FIG. 5D).
The SP2 sub-pocket refers to a pocket formed by residues W86, S129, W227 and F311 in AKR1C3. This pocket is where the 8α-chain of PGD2 and the 12β-chain of bimatoprost were bound (Komoto et al., 2004, Biochemistry 43:2188-98). It appears that this sub-pocket is only used by the two prostanoids.
The SP3 sub-pocket refers to a large pocket lined by residues Y24, E192, S217, S221, Q222, Y305, and F306 in AKR1C3. These residues surround the indole ring and the carboxylate group of the indomethacin molecule at pH 6.0. SP3 is not occupied by other ligands in AKR1C3.
The variety of AKR1C3 ligands in structure and size demonstrate the flexibility of the enzyme's ligand binding site. The key residues to AKR1C3's ability to accommodate different ligands are W227, F306, and F311. These residues can assume different conformations and result in “induced-fit” to various ligands. W227 controls the sizes of SP2 and the steroid channel. F306 lines the SP1 site for the AKR1C3-NADP+-flufenamic acid complex, but assumes a different rotamer conformation in the crystal of the AKR1C3-NADP+-indomethacin complex, thereby exposing the SP3 pocket. F311 forms part of SP1 for the binding of flufenamic acid, but lines SP2 for the binding of bimatoprost and PGD2.
Comparison of the ligand binding sites of AKR1C1-4 reveals that there are considerable structural differences in sub-pockets between AKR1C3 and the other isoforms (Couture et al., 2003, J. Mol. Biol. 331:593-604). SP1 is significantly larger for AKR1C3. The difference in the size of SP1 is largely due to the shift in main chain positions of residues 305-311 between AKR1C3 and the other enzymes (FIG. 6A). As a result, residue 308 collapses inward and significantly reduces the size of SP1 for AKR1C1, AKR1C2 and AKR1C4. In AKR1C3 the side chain of S308 is not involved in ligand binding (>4 A), but the corresponding residues of L308 in AKR1C2 and M308 in AKR1C4 would extend into SP1. In addition, different residues at positions 118 and 319 also reduce the size of SP1 in AKR1C ½ and AKR1C4. In AKR1C3, the side-chains of S118 and Y319 are >4 Å away from the ligand, whereas the corresponding residues of F118 (1.7 Å) and F319 (˜1 Å) in AKR1C2 would clash with a long ligand such as PGD2 or bimatoprost (Komoto et al., 2006, Biochem. 45:1987-96). Importantly, the serine residue at position 118 in AKR1C3 is capable of forming hydrogen-bonding interactions with a ligand, i.e., with the amide group of the 8α-chain of bimatoprost (Komoto et al., 2006, Biochem. 45:1987-96). In contrast, the corresponding residue for AKR1C1, AKR1C2 and AKR1C4 is a phenylalanine incapable of hydrogen bonding with a ligand.
No ligands have been observed to occupy the SP2 site in crystals of AKR1C1, AKR1C2, or AKR1C4. Comparison of the SP2 sites of these enzymes shows structural differences at position 129 and 311 that make the SP2 pocket shorter for AKR1C1, AKR1C2, and AKR1C4 than that for AKR1C3 (FIG. 6B). In addition, S129 of AKR1C3 can form hydrogen-bonding interactions with a ligand, i.e., with the carboxylate group of the 8α-chain of PGD2 or with the hydroxyl group on 12β-chain of bimatoprost (Komoto et al., 2006, Biochem. 45:1987-96). In contrast, the corresponding residues in other isoforms—I129 for AKR1C1 and AKR1C2 and L129 for AKR1C4—are incapable of hydrogen bonding with a ligand.
Structural differences at position 306 also cause the SP3 site of AKR1C3 to differ from those for the other enzymes (FIG. 6C). In the AKR1C3-NADP+-indomethacin complex at pH 6.0, the side chain of F306 assumes a conformation that points away from the ligand. However, the corresponding residues L306 of AKR1C1 and AKR1C2 and V306 of AKR1C4 have more rigid side chains that would clash with the indole ring of indomethacin.
Despite the availability of structural information, there has been no report to date of an AKR1C3 inhibitor that does not inhibit AKR1C1 and AKR1C2 and has good developmental properties. Such a selective inhibitor is needed to explore the role of AKR1C3 in normal and aberrant cell signaling. Such a selective compound may find use in treating CRPC through the inhibition of AKR1C3. There is thus a need in the art to identify novel inhibitors of AKR1C3 that show selectivity over AKR1C1 or AKR1C2. The present invention fulfills this need.